The objective of the proposed research is to characterize the RNA splicing reaction by which intervening sequences are removed from RNA molecules in eukaryotic organisms. In the amplified ribosomal RNA genes of the ciliated protozoan Tetrahymena thermophila, the 26S rRNA coding region is interrupted by a 400 base pair intervening sequence. This sequence is contained in the primary transcript but is subsequently deleted from the RNA by splicing. We have recently developed an in vitro splicing system that permits the isolation of the excised 400 nucleotide RNA fragment ("IVS RNA"). The splicing reaction will be characterized in vitro in isolated nuclei as well as in vivo to determine the nature of the reaction products and the kinetics of their synthesis and degradation. Hybridization of nuclear RNA with recombinant DNA containing the intervening sequence will be used to isolate the unspliced pre-rRNA. This material will provide the basis for an assay to detect splicing activity, which should allow purification of the enzyme(s) involved. The enzyme(s) may require an rRNA-protein complex as a substrate, in which case nascent ribosomal particles will be isolated. The number of different enzymes involved in the pre-rRNA splicing and the specificity of each enzyme will be investigated.